Project Summary CD4+ T cells (TCD4+) recognize antigens in the form of peptides (epitopes) bound to cell surface MHC class II molecules (MHC-II) and are critical for the development of protection against most pathogens. Decades of work with nominal protein antigens has produced a standard model of peptide generation and presentation: Internalized antigen is digested and loaded onto nascent MHC-II-bound peptides in a late endosomal compartment followed by transit of the resulting complexes to the cell surface. In recent years, as work has extended to more complex antigens, it has become apparent that there are multiple alternatives to this classical ?exogenous? pathway, including a poorly understood ?endogenous? processing ?network? comprised of antigen processing machinery beyond the endocytic compartment and a transfer pathway in which antigen is conveyed from the infected APC to an uninfected APC. Indeed, our recent work with influenza has demonstrated that epitopes produced by the endogenous MHCII processing network drives the vast majority of the TCD4+ response to an influenza infection, while those produced by antigen transfer make a minor contribution. Here we propose to study the roles of these alternative mechanisms in TCD4+ responses to Salmonella enterica serovar Typhimurium (S. Typhimurium), which injects a number of proteins into the host cell cytosol as part of the infection program. Based upon our recent work and that of others, we hypothesize that: 1) TCD4+ responses to S. Typhimurium are focused upon these secreted proteins (SPs), and 2) SPs are processed primarily by the endogenous network and secondarily by antigen transfer, the contribution of the former being dampened by the Salmonella-encoded SteD protein, which down-modulates MHC-II levels in the infected cell. In Aim 1 we will assess the relative contributions to the global TCD4+ of SPs, vs. 2 classes of structural proteins (cytosolic and fimbrial) and the influence of SteD on this balance. Subsequently, we will explore the importance of delivery to the host cell cytosol for efficient epitope generation. Complementing this approach, in Aim 2, through the use of knockout mice, knockdown strategies, and chemical inhibitors we will identify in vivo and in vitro the cellular components that contribute to the generation of individual S. Typhimurium epitopes from the three classes of protein. Together, these Aims will elucidate the origins of S. Typhimurium epitopes, and the processing pathways that facilitate their presentation to TCD4+ cells. Our expectation is that the outcomes of this exploratory R21 project will: 1) further guide the field toward a more complex view of MHC-II processing and presentation, 2) reveal an elegantly simple system for distinguishing between pathogenic and non-pathogenic bacteria, and 3) provide key insights into the rational development of effective vaccines against bacterial pathogens, a high priority considering the recent and alarming developments with respect to antibiotic resistance, and an area we intend to contribute to in building upon the proposed project.